Analytical Sciences

Microfluidics-based in situ Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell

Arisa KURODA,* Yuri ISHIGAKI,* Mats NILSSON,** Kiichi SATO,*** and Kae SATO*

*Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, Bunkyo, Tokyo 112-8681, Japan
**Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Box 1031, Se-171 21 Solna, Sweden
***Division of Molecular Science, School of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan

In situ padlock/rolling circle amplification (RCA) is a method used to amplify, visualize, and quantify target DNA molecules in cells. However, the multiple reaction steps involved make this technique costly and cumbersome. We developed a novel, simplified, automated microfluidic system for RCA, and demonstrated its effectiveness by counting amplified mitochondrial DNA fragments in HeLa cells. After optimizing the volume of the reaction solutions and washing buffer composition, the product yield was equal to that obtained by the conventional manual method. The required volume of reagents was reduced to 10 μL, which is less than half the volume used in the conventional method. To the best of our knowledge, this is the first report of an automated microfluidic method for in situ padlock/RCA, which can be useful for making highly efficient pathological diagnoses.

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